Mesenchymal stem cells (“MSC” or “MSCs”) can be found in bone marrow, blood, dermis, periosteum and other tissues of the body, and are capable of differentiating into a variety of cell types, including adipose, areolar, osseous, cartilaginous, elastic, marrow stroma, muscle, fibrous connective tissue, and cardiac tissue, depending upon various in vivo or in vitro factors and influences. Such cells are disclosed, for example, in U.S. Pat. Nos. 5,197,985; 5,226,914; 5,486,359; 5,837,539, and 6,087,113, each of which are independently incorporated by reference in their entirety.
MSCs have been shown to engraft and selectively differentiate, based on the tissue environment, to lineages such as muscle, bone, cartilage, marrow stroma, tendon and fat. Due to their cellular origin and phenotype, these cells do not provoke an adverse immune response, allowing for the development of products derived from unrelated human donors.
In general, MSCs are isolated from the tissue from which they are obtained, purified, and then expanded in an appropriate culture medium. The culture medium contains a variety of components that support the expansion of the MSCs, such as serum, which comprises serum proteins (e.g., serum albumin, such as bovine serum albumin); growth factors; and cytokines. After isolation, purification, and culture expansion, the MSCs are subjected to a series of washes and, optionally, centrifugation. The MSCs then may be frozen and stored in an appropriate cryopreservation medium, for example a cryopreservation medium comprising dimethyl sulfoxide (“DMSO”). Subsequently, the MSCs are thawed just prior to administration to a patient.
The manufacturing process for the expansion of MSCs involves cell culturing in the presence of non-autologous serum and cell harvesting by non-autologous trypsin; in some processes, the non-autologous serum is fetal bovine serum (“FBS”) and the non-autologous trypsin is porcine trypsin. The ex vivo expansion of human MSCs (“hMSCs”) using animal reagents leads to the presence of residual macro-molecules of non-human origin (for example, macro-molecules of porcine and bovine origin) in the ultimate product. After expanding hMSCs in media comprising non-human products, an increased amount of xenogeneic substances can be observed relative to hMSCs expanded in media comprising human products.
Bovine serum albumin (“BSA”) is a significant component of FBS. Both BSA and porcine trypsin are known allergens. As such, they can trigger adverse reactions in patients susceptible to bovine and porcine macro-molecules, and can cause non-allergic patient sensitization leading to allergic reactions upon multiple exposures (See, e.g., Cotten HR et al., N Engl J Med, 1975, 292:1050; Moneret-Vautrin A. et al., Allergy, 1991, 46:228; Orta M et al., Ann Allergy Asthma Immunol 2003, 90:446; de Benito V. et al., Allergologia et Immunopathologia, 2001, 29:272). Increased amounts of FBS present in culturally expanded MSCs also may induce undesired side effects in patients, such as undesirable immune responses, pulmonary embolism, vasoconstriction, cardiac shock, or death. The presence of residual BSA or porcine trypsin may increase immunogenicity and accelerate clearance or elimination of MSCs from the recipient. Increased amounts of FBS present in pharmaceutical compositions comprising culturally expanded MSCs can increase the risk of transmitting viruses, prion diseases, and xenogeneic proteins to patients receiving such MSC-based therapies. Increased amounts of FBS, particularly BSA, present in pharmaceutical compositions comprising culture-expanded hMSCs may initiate immune responses against these xenogeneic substances. For example, if the MSC preparation administered to a patient contains BSA or other xenogeneic proteins, such xenogeneic proteins may trigger an undesirable immune response. Xenogeneic proteins may elicit cell-mediated or humoral immune responses (e.g., the generation of anti-bovine serum protein antibodies), which may result in less efficient engraftment of the MSCs, particularly if such xenogeneic proteins become associated with MSC cell-surface membranes. As such, a new approach is needed to reduce the amount of xenogeneic substances, including FBS, and particularly BSA, present in pharmaceutical compositions comprising culturally expanded MSCs. A new approach is needed to reduce the amount of xenogeneic substances, including sugars, proteins and other macromolecules present in culture expanded MSCs, which could increase the safety profile of the resultant MSC composition.
Media comprising alternative sera such as autologous human serum have been proposed, however, the use of autologous serum is not possible when the quantities of cells required in the ultimate MSC product exceed that which can be grown in a fixed amount of autologous serum. Additionally, the use of autologous human serum presupposes that the patient will have sufficient time and be in sufficient health to donate serum in advance of the initiation of MSC therapy. The current conventional MSC culturing process typically requires 2 to 10 weeks to isolate, expand, harvest and purify a suitable number of cells to constitute a pharmaceutical treatment. In some cases, a pharmaceutical treatment consists of 1 dose. In other cases, a pharmaceutical treatment consists of 2 or more doses. Unfortunately, in some cases, MSC therapy is needed less than about 2 weeks from diagnosis or presentation of clinical symptoms, or in less than about 1 week from diagnosis or presentation of clinical symptoms, or in less than about 48 hours of diagnosis or presentation of clinical symptoms. When MSC therapy is needed within a short time period from diagnosis or presentation of clinical symptoms, hMSCs that have already been manufactured, purified and cryopreserved exhibit the significant benefit of being available upon diagnosis or presentation of an acute illness.
Moreover, human serum, including autologous human serum, exhibits a statistically significant increase in the risk of transmitting a disease, for example, a viral disease, to the recipient of the MSC pharmaceutical composition.
Spees et al., mention combinations of media comprising serial passages in fetal calf serum (“FCS”) and autologous human serum. (Spees et al., Mol Therapy, 2004, 9: 747). Final compositions produced by serial combinations of media yielded greater than a 15-fold range in residual FCS per sample according to SDS-Page electrophoresis of labeled FCS after 50 wash cycles. Protocols requiring autologous human serum and extensive washing that do not provide more reproducible final compositions are academically interesting, but do not provide the quality or consistency required to manufacture a pharmaceutical composition suitable for administration to a human.
Risk doses and thresholds for clinical reactivity among allergic patients have been established for a number of antigens. (Moneret-Vautrin A. & Kanny G., Curr Opin Allergy Clin Immunol, 2004, 4:215; Bindslev-Jensen C et al., Allergy, 2002, 57:741). Though these thresholds are established for oral administration of antigens, thresholds for intravenous (“IV”) exposure to allergens are unknown. (Wensing M. et al., J Allergy Clin Immunol, 2002, 110:915; Taylor SL et al., Clin Exp Allergy, 2004, 34:689). Therapeutic decisions regarding IV administration of compositions comprising MSCs are complicated by the absence of threshold data and reports in the literature showing that cellular and animal-derived products may cause serious adverse reactions (for example, anaphylaxis and serum sickness-like disease). (Moneret-Vautrin A et al., Allergy, 1991, 46:228; Orta M et al., Ann Allergy Asthma Immunol 2003, 90:446; de Benito V. et al., Allergologia et lmmunopathologia, 2001, 29:272).
As an example, risk doses and thresholds for clinical reactivity among allergic patients are established for a number of antigens, most of which relate to food allergen categories (Moneret-Vautrin A. & Kanny G., Curr Opin Allergy Clin Immunol, 2004, 4:21). Because these thresholds were established for oral administration of antigens, they are expected to be different from thresholds for IV administration. Again, thresholds for IV exposure to allergens remain unknown. (Taylor SL et al. Clin Exp Allergy, 2004, 34:689). The absence of threshold data and reports in the literature showing that cellular and animal-derived products may cause serious adverse reactions (for example, anaphylaxis and serum sickness like disease) exclude use of therapeutics manufactured in the presence of bovine or porcine products. (Orta M. et al., Ann Allergy Asthma Immunol 2003, 90:446).
Perotti et al mention centrifugal filtration as a technique useful for removing the cryopreservative DMSO from umbilical cord blood. (Perotti CG et al., Transfusion, 2004, 44(6):900-906). Calmels et al. mention centrifugal filtration as a technique useful for removing DMSO from hematopoietic stem cell grafts. (Calmels B et al., Bone Marrow Transplant., 2003, 31(9):823-828). Hampson et al. mention methods to wash cultured bone marrow mononuclear cells. (US 2008/0175825). Post-wash residual BSA levels from the cell culture supernatant were reported to be about less than 3 μg/ml. Using a Cytomate instrument to wash bone marrow mononuclear cells, Hampson et al. obtained about 70% cell viability post-wash. Hampson et al. indicated that this significant drop in cell viability may have been due to cellular damage caused by mechanical forces applied during the process.
Protocols requiring extensive washing of cells do not provide the quality or consistency required to manufacture a pharmaceutical composition suitable for administration to a human. Furthermore, the effects of extensive washing protocols on the viability of cells and the efficacy a pharmaceutical composition comprising such cells is unknown.
Additionally, many published purification protocols comprise at least one step involving transfer of the MSC-containing intermediate product where the product is exposed to the external environment (i.e. not a closed system). As closed systems carefully control the quantity and quality of materials entering and leaving the system, as well as the manner by which these materials enter or leave, the development of a closed manufacturing system for the preparation of MSC pharmaceutical compositions would represent a significant accomplishment in the art.
With these challenges in mind, it is necessary to: 1) establish a threshold dose for residual components in the product that will minimize risk of allergic reactions in patients; 2) provide a method for purifying an hMSC composition to reduce the amount of residual components, including allergens, below the threshold level, while minimizing cellular damage and maintaining cell viability; and, 3) provide an hMSC composition comprising less than the threshold amount of residual components, including allergens, limited cellular damage, and a high proportion of viable cells.
In summary, the state of the art related to methods of preparing pharmaceutical MSC compositions comprises one significant long felt need: reducing the immunogenicity of MSC compositions cultured in non-human serum. Further, the present technology described and claimed herein surprisingly identified a challenge that had not been previously recognized in the conventional art as a significant shortcoming: reducing the extent of MSC aggregation.